Methods for treating bipolar disorder

ABSTRACT

The invention is directed to a method of treating bipolar disorder in a subject, comprising administering a therapeutically effective amount of a carbamate compound, or pharmaceutically acceptable salt or amide thereof.

BACKGROUND OF THE INVENTION

The present invention relates a method of treating bipolar disorder.More specifically, the present invention is directed to a method ofusing a carbamate compound alone or in combination with othermedications, for the treatment of bipolar disorder.

DESCRIPTION OF RELATED ART

Bipolar Disorder (BPD) is a chronic and cyclic psychiatric disordercharacterized by mania and depression. Bipolar I disorder is qualifiedby at least one lifetime manic or mixed episode (Miklowitz & Johnson,2009). Mania must last at least one week or require hospitalization andsymptoms during this period include irritability, euphoria, decreasedneed for sleep, grandiose ideas, impulsive behavior, increasedtalkativeness, racing thoughts, increased activity and distractibility.A mixed episode includes manic symptoms along with a simultaneousdepression. Bipolar II consists of periods of hypomania and majordepression. Hypomania is similar to mania but considered milder in thatthe patient is not socially or vocationally impaired but shows changesin functioning (Miller et al., 2009). During major depression, thepatient experiences intense feelings of sadness/worthlessness, loss ofinterests, fatigue, insomnia, agitation, weight gain/loss and suicidalideation/attempts. Lifetime prevalence rate is estimated atapproximately 4% based upon the National Comorbidity Survey replication(Kessler et al., 2005a). There is high comorbidity of bipolar disorderwith attention hyperactivity disorder and substance abuse among others(Kessler et al., 2005b).

Treatment of bipolar disorder is aimed at treating the manic, hypomanicand depressive symptoms of the disorder as well, as at maintainingtreatment by reducing or preventing the cyclicity of the disorder.Lithium has long been considered the standard treatment of bipolardisorder based on a few early clinical trials which have since beenhighly criticized for methodological issues (Vieta & Rosa, 2007).Nevertheless, lithium unquestionably is efficacious for mania but lessso in treating depression (Frye et al., 2004). In addition, patientstreated with lithium can show some signs of illness, abrupt cessation oftreatment can induce a manic episode and further not all can toleratethe treatment (Vieta & Rosa, 2007). Carbamazepine and valproate may alsobe beneficial for BPD but additional long-term clinical studies arenecessary (Azorin & Kaladjian, 2009). Second generation antipsychoticsincluding olanzapine, quetiapine, risperidone and clozapine have shownsome promise in the treatment of BPD but side effects includingsedation, weight gain and metabolic disorders are common ('Vista & Rosa,2007). Antidepressants, fluoxetine, paroxetine and venlafaxine show someefficacy in the treatment of bipolar depression (Hard & Levkovitz, 2008;Azorin & Kaladjian, 2009). However, switch rate defined as the changefrom depression to mania or hypomania is reported to be increased forantidepressants especially venlafaxine but there was no consistency inwhat constituted a switch and how dropouts from placebo groups werehandled. Cycle acceleration or rapid cycling is another risk associatedwith antidepressant treatment in BPD (Harel & Levkovitz, 2008). Similarto switch rate, there a lack of a consistent definition and controlleddata on cycle acceleration. Thus, more controlled studies with,systematic and objective measures are needed to adequately assess theincidence of these risks.

There appears to be an implication of dopamine in bipolar disorder(Cuellar et al., 2005; Cousins et al., 2009; Rapoport et al., 2009).While the role is complex, the use of stimulants such as amphetaminetends to mimic bouts of mania. On the other hand, dopamine agonistsalleviate the apparent dopamine deficiency observed in Parkinson'sdisease-induced depression. Untreated bipolar depressive patients showeddecreased levels of the dopamine metabolite, homovanillic acid (HVA), inthe cerebrospinal fluid. Dopaminergic agents should be sought to providea balance in the dopaminergic neurosystems in order to prevent theextreme cycling between mania and depression in BPD. There also has beenreported to be an imbalance of norepinephrine in bipolar disorder(Cueller et al., 2005).

In a survey of psychiatrists in the UK and US, high unmet, needs in BPDwere identified (Chengappa & Williams, 2005). Underserved subsets of BPDwere those patients who are rapid cyclers and those with comorbidsubstance abuse. It was felt that BPD would benefit by treatment thatwas better tolerated and effective in all phases of the disease withreduced relapse frequency and a fast onset of action.

Accordingly, there is a need manic and depressive episodes and othercomorbidities including attention-hyperactivity disorder and substanceabuse and reduce side effect profiles.

SUMMARY OF THE INVENTION

The present invention is directed to a method of treating bipolardisorder comprising the administration of a therapeutically effectiveamount of a compound having structural Formula (1) or a pharmaceuticallyacceptable salt thereof, to a mammal in need of the treatment:

wherein,

R is selected from the group consisting of hydrogen, lower alkyl of 1 to8 carbon atoms, halogen selected from F, Cl, Br and I, alkoxy of 1 to 3carbon atoms, nitro group, hydroxy, trifluoromethyl, and thioalkoxy of 1to 3 carbon atoms;

x represents the number of R, and, is an integer of 1 to 3, with theproviso that R may be the same or different when x is 2 or 3;

R₁ and R₂ can be the same or different from each other and areindependently selected from the group consisting of hydrogen, loweralkyl of 1 to 8 carbon atoms, aryl of 3 to 7 carbon atoms, arylalkyl of3 to 7 carbon atoms, and cycloalkyl of 3 to 7 carbon atoms;

R₁ and R₂ can be joined to form a 5 to 7-membered heterocyclesubstituted with a member selected from the group consisting ofhydrogen, alkyl of 1 to 8 carbon atoms, and aryl of 3 to 7 carbon atoms,wherein the heterocyclic compound comprises 1 to 2 nitrogen atoms and 0to 1 oxygen atom, and the nitrogen atoms are not directly connected witheach other or with the oxygen atom.

In another embodiment, the present invention provides a method ofimproving symptoms associated with bipolar disorder in a subject,comprising the step of the administration, to a subject in need of suchtreatment, of a therapeutically effective amount a compound of theFormula (1) or a pharmaceutically acceptable salt thereof.

In further embodiment, the present invention provides a method ofameliorating or eliminating symptoms of bipolar <disorder in a subject,comprising the step of the administration, to a subject in need of suchtreatment, of a therapeutically effective amount a compound of theFormula (1) or a pharmaceutically acceptable salt thereof.

In additional embodiment, the present invention is directed to apharmaceutical composition for treating bipolar disorder comprising acompound of the Formula (1) or a pharmaceutically acceptable saltthereof, as an active ingredient.

In another embodiment, the present invention provides a pharmaceuticalcomposition for improving symptoms associated with bipolar disorder in asubject, comprising a compound of the Formula (1) or a pharmaceuticallyacceptable salt thereof, as an active ingredient.

In further embodiment, the present invention, provides a pharmaceuticalcomposition for ameliorating or eliminating symptoms of bipolar disorderin a subject, comprising a therapeutically effective amount a compoundof the Formula (1) or a pharmaceutically acceptable salt thereof.

The compound having structural Formula (1) is an enantiomersubstantially free of other enantiomers or an enantiomeric mixturewherein one enantiomer of the compound having structural Formula (1)predominates. One enantiomer predominates to the extent of about 90% orgreater, and preferably about 98% or greater.

The enantiomer is (S) or (L) enantiomer as represented by StrueturalFormula (1a) or (R) or (D) enantiomer, as represented by StructuralFormula (1b):

Preferably, R, R₁ and R₂ are all selected from hydrogen, which are shownin the following formula:

Embodiments of the invention include a method for using the enantiomerof Formula 1 substantially free of other enantiomers that is theenantiomer of Formula 1b or an enantiomeric mixture wherein theenantiomer of Formula 1b predominates. (Note: in the structural formulaof Formula 1b below the amino group attached to the beta carbon projectsinto the plane of the paper. This is the dextrorotary (D) enantiomerthat is of absolute configuration (R).)

DETAILED DESCRIPTION OF THE EMBODIMENT

These and other objects of the invention will be more fully understoodfrom the following description of the invention and the claims appendedhereto.

The present invention is directed to a method of treating bipolardisorder comprising the administration of a therapeutically effectiveamount of a compound having structural Formula (1) or enantiomers,diastereomers, racemates or mixtures thereof, or hydrates, solvates andpharmaceutically acceptable salts and amides thereof; to a mammal inneed of the treatment:

wherein,

R is selected from the group consisting of hydrogen, lower alkyl of 1 to8 carbon atoms, halogen selected from F, Cl, Br and I, alkoxy of 1 to 3carbon atoms, nitro group, hydroxy, trifluoromethyl, and thioalkoxy of 1to 3 carbon atoms;

x represents the number of R, and is an integer of 1 to 3, with theproviso that R may be the same or different when x is 2 or 3;

R₁ and R₂ can be the same, or different from each other and areindependently selected from the group consisting of hydrogen, loweralkyl of 1 to 8 carbon atoms, aryl of 3 to 7 carbon atoms, arylalkyl of3 to 7 carbon atoms, and cycloalkyl of 3 to 7 carbon atoms;

R₁ and R₂ can be joined to form a 5 to 7-membered heterocyclesubstituted with a member selected from the group consisting ofhydrogen, alkyl of 1 to 8 carbon atoms, and aryl of 3 to 7 carbon atoms,wherein the heterocyclic compound comprises 1 to 2 nitrogen atoms and 0to 1 oxygen atom, and the nitrogen atoms are not directly connected witheach other or with the oxygen atom.

The present method also includes the use of a compound selected from thegroup consisting Formula 1a or 1b, or enantiomers, diastereomers,racemates or mixtures thereof, or hydrates, solvates andpharmaceutically acceptable salts and amides thereof:

wherein Rx, R₁ and R₂ are the same as defined above.

The present method also preferably includes the use of the D (ordextrorotary) enantiomer (of absolute configuration R) selected from thegroup consisting of a compound of Formula 1 or an enantiomeric mixturethereof. In the structural formula of Formula 1b, the amino groupattached to the beta carbon projects into the plane of the paper. Thisis the dextrorotary (D) enantiomer that is of absolute configuration(R).

Preferably, in the Structural Formula 1, R, R₁ and R₂ are hydrogen asrepresented by following Structural Formula:

O-carbamoyl-(D)-phenylalaninol is also named(R)-(beta-amino-benzenepropyl) carbamate monohydrochloric acid. Forenantiomeric mixtures, wherein O-carbamoyl-(D)-phenylalaninolpredominates, preferably, to the extent of about 90% or greater, andmore preferably about 98% or greater.

The compounds of Formula 1 can be synthesized by methods known to askilled person in the art. Some reaction schemes for synthesizingcompounds of Formula (1) have been described in published; U.S. Pat. No.5,705,640, U.S. Pat. No. 5,756,817, U.S. Pat. No. 5,955,499, and U.S.Pat. No. 6,140,532. Details of the above reactions schemes as well asrepresentative examples on the preparation of specific compounds havebeen described m published; U.S. Pat. No. 5,705,640, U.S. Pat. No.5,756,817, U.S. Pat. No. 5,955,499, U.S. Pat. No. 6,140,532, allincorporated herein by reference in their entirety.

The salts and amides of the compounds of Formula (1) can be produced bytreating the compound with an acid (HX) in suitable solvent or by meanswell known to those of skill in the art.

From Structural Formula 1, it is evident that some of the compounds ofthe invention have at least one and possibly more asymmetric carbonatoms. It is intended that the present invention include within itsscope the stereochemically pure isomeric forms of the compounds as wellas their racemates. Stereochemically pure isomeric forms may be obtainedby the application of art known principles. Diastereoisomers may beseparated by physical separation methods such as fractionalcrystallization and chromatographic techniques, and enantiomers may beseparated from each other by the selective crystallization of thediastereomeric salts with optically active acids or bases or by chiralchromatography. Pure stereoisomers may also be prepared syntheticallyfrom appropriate stereochemically pure starting materials, or by usingstereoselective reactions.

During any of the processes for preparation of the compounds of thepresent invention, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This maybe achieved by means of conventional protecting groups, such as thosedescribed in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie,Plenum Press, 1973; and T.W. Greene & P.G.M. Wuts, Protective Groups inOrganic Synthesis, Third Edition, John Wiley & Sons, 1999 The protectinggroups may be removed at a convenient subsequent stage using methodsknown from the art.

The present invention is based in part on the discovery thatphenylalkylamino carbamates of Formula 1 discussed above have novel andunique pharmacological properties. These compounds have been shown inseveral animal models to have the ability to treat bipolar disorder andimprove, ameliorate or eliminate symptoms associated with bipolardisorder.

Although the precise mechanism of action is not completely understood,it is known that these compounds do not work by the same mechanisms asmost other known treatments for bipolar disorder. For these reasons, thecompounds of Formula 1 are especially suitable for use as sole oradjunctive treatment for bipolar disorder and improvement, ameliorationor elimination of symptoms associated with bipolar disorder.

Thus, these compounds can be safely used alone or in combination withother useful medications to provide enhanced efficacy and reduced sideeffects because smaller doses of each drug that could be used.

In one aspect, this invention relates to methods to treat bipolardisorder; the method comprising administering to a subject sufferingfrom bipolar disorder a therapeutically effective amount of one or moreof the carbamate compounds of the invention or a pharmaceuticallyacceptable, salt or amides thereof, optionally with a pharmaceuticallyacceptable carrier, diluent or excipient. The method may furthercomprise the step of identifying a subject suffering from bipolardisorder before the administering step.

In another aspect, this invention also provides, a method fordiminishing, inhibiting or eliminating the symptoms associated withbipolar disorder including manic (e.g., unrealistic plans, spendingsprees, recklessness, decreased need for sleep, pressured speech,inflated self-esteem, uncharacteristically poor judgment, indifferent topersonal grooming and unusual sexual drive) and depressive (sadness andcrying spells, hypersomnolence insomnia, anorexia, excessive eating andmay gain weight, loneliness, self-loathing, apathy or indifference,depersonalization, loss of interest in sexual activity, shyness orsocial anxiety, chronic pain, lack of motivation, morbid/suicidalideation, irritability or restlessness) symptoms in a subject sufferingfrom bipolar disorder which comprises administering to the subject aneffective amount of carbamate compounds of the invention, apharmaceutically acceptable salt, or amides thereof to diminish, inhibitor eliminate said symptoms.

Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

It is to be understood that this invention is not limited to theparticular methodology, protocols, animal species or genera, andreagents described, as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention that will be limited only by the appended claims.

As used herein the term “subject”, refers to an animal, preferably amammal, and most preferably a human both male and female, who has beenthe object of treatment, observation or experiment.

The term “therapeutically effective amount” as used herein, means thatamount of active compound or pharmaceutical agent that elicits thebiological or medicinal response in a tissue system, animal or humanthat is being sought by a researcher, veterinarian, medical doctor orother clinician, which includes alleviation of one or more of the signsor symptoms of the disease or disorder being treated.

The term “prophylactically effective amount” is intended to mean thatamount of a pharmaceutical drug that will prevent or reduce the risk ofoccurrence of the biological or medical event that is sought to beprevented of a tissue, a system, animal or human that is being sought bya researcher, veterinarian, medical doctor or other clinician.

The term “pharmaceutically acceptable salts or amides” shall meannon-toxic salts or amides of the compounds employed in this inventionwhich are generally prepared by reacting the free acid with a suitableorganic or inorganic base. Examples of such salts include, but are notlimited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate,bitartrate, borate, bromide, calcium, calcium edetate, camsylate,carbonate, chloride, clavulanate, citrate, dihydrochloride: edetate,edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate,lactate, Iactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,oleate, oxalate, pamaote, palmitate, panthothenate,phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium,stearate, subacetate, succinate, militate, tartrate, teoclate, tosylate,triethiodide, valerate.

Therefore, the term “a patient in need of treatment” as used herein willrefer to any subject or patient who currently has or may develop any ofthe above syndromes or disorders, including any mood disorder which canbe treated by antidepressant medication, or any other disorder in whichthe patient's present clinical condition or prognosis could benefit fromthe administration of one or more compounds of Formula (1) alone or incombination with another therapeutic intervention including but notlimited to another medication.

The term “treating” or “treatment” as used herein, refers to any indiciaof success in, the prevention or amelioration of an injury, pathology orcondition of bipolar disorder and modification or symptoms of bipolardisorder, including any objective or subjective parameter such asabatement; remission; diminishing of symptoms or making the injury,pathology, or condition more tolerable to the patient; slowing in therate of degeneration or decline or worsening of the illness; making thefinal point of worsening less debilitating; or improving a subject'sphysical or mental well-being. The treatment or amelioration of symptomscan be based on objective or subjective parameters; including theresults of a physical examination, neurological examination, and/orpsychiatric evaluations. Accordingly, the term “treating” or “treatment”includes the administration of the compounds or agents of the presentinvention, for treatment of any form of bipolar disorder in both malesand females. In some instances, treatment with the compounds of thepresent mention will done in combination with other compounds toprevent, inhibit, or arrest the progression of bipolar disorder.

The term “therapeutic effect” as used herein, refers to the effectiveimprovement in or reduction of symptoms of bipolar disorder. The term “atherapeutically effective amount” as used herein means, a sufficientamount of one or more of the compounds of the invention to produce atherapeutic effect, as defined above, in a subject or patient in need ofsuch bipolar disorder treatment.

The terms “subject” or “patient” are used herein interchangeably and asused herein mean any mammal including but not limited to human beingsincluding a human patient or subject to which the compositions of theinvention can be administered. The term mammals include human patients,both male and female and non-human primates, as well as experimentalanimals such as rabbits, rats, mice, and other animals.

Methods are known in the art for determining therapeutically andprophylactically effective doses for the instant pharmaceuticalcomposition. For example, the compound can be employed at a daily dosein the range or about 0.1 mg to 400 mg usually on a regimen of severaltimes, for example, 1 to 2 times per day, for an average adult human.The effective amount, however, may be varied depending upon theparticular compound used, the mode of administration, the strength ofthe preparation, the mode of administration, and the advancement of thedisease condition. In addition, factors associated with the particularpatient being treated, including patient age, weight, diet and time ofadministration, will result in the need to adjust dosages.

The compound may be administered to a subject, by any conventional routeof administration, including, but not limited to, intravenous, oral,subcutaneous, intramuscular, intradermal and parenteral. Depending onthe route of administration, compounds of Formula (1) can be constitutedinto any form. For example, forms suitable for oral administrationinclude solid forms, such as pills, gelcaps, tablets, caplets, capsules(each including immediate release, timed release and sustained releaseformulations), granules, and powders. Forms suitable for oraladministration also include liquid forms, such as solutions, syrups,elixirs, emulsions, and suspensions, la addition, forms useful forparenteral administration include sterile solutions, emulsions andsuspensions.

To prepare the pharmaceutical compositions of this invention, one ormore compounds of formula (1) or salt thereof as the active ingredientis intimately admixed with a pharmaceutical carrier according toconventional pharmaceutical compounding techniques. Carriers arenecessary and inert pharmaceutical excipients, including, but notlimited to, binders, suspending agents, lubricants, flavorings,sweeteners, preservatives, dyes, and coatings. In preparing compositionsin oral dosage form, any of the usual pharmaceutical carriers may beemployed. For example, for liquid oral preparations, suitable carriersand additives include water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like; for solid oralpreparations, suitable carriers and additives include starches, sugars,diluents, granulating agents, lubricants, binders, disintegrating agentsand the Re. For parenteral use, the carrier will usually comprisesterile water, though other ingredients, for example, for purposes suchas aiding solubility or for preservation, may be included. Injectablesuspensions may also be prepared, in which case appropriate liquidcarriers, suspending agents and the like may be employed.

Because of their ease in administration, tablets and capsules representthe most advantageous oral dosage unit form, in which case solidpharmaceutical carriers are obviously employed. If desired, tablets maybe sugar coated or enteric coated by standard techniques. Suppositoriesmay be prepared, in which case cocoa butter could be used as thecarrier. The tablets or pills can be coated or otherwise compounded toprovide a dosage form affording the advantage of prolonged action. Forexample, the tablet or pills can comprise an inner dosage and an outerdosage component, the latter being in the form of an envelope over, theformer. The two components can be separated by an enteric layer, whichserves to resist disintegration in the stomach and permits the innercomponent to pass intact into the duodenum or to be delayed in release.A variety of material can be used for such enteric layers or coatings,such materials including a number of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

The active drug can also be administered in the form or liposomedelivery systems, such as small unilamellar vesicles, large urilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

Active drag may also be delivered by the use of monoclonal antibodies asindividual carriers to which the compound molecules are coupled. Activedrug may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinyl-pyrrolidone, pyrancopolymer, polyhydroxy-propyl-methacrylamide-phenol,polyhydroxy-ethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, active drug may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polyglycolicacid, copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross linked or amphipathicblock copolymers of hydrogels.

Preferably these compositions are in unit dosage forms, such as tablets,pills, capsules, powders, granules, sterile parenteral solutions orsuspensions, metered aerosol or liquid sprays, drops, ampoules,auto-injector devices or suppositories, for oral parenteral, intranasal,sublingual or rectal administration, or for administration by inhalationor insufflation.

Alternatively, the composition may be presented in a form suitable foronce-weekly or once-monthly administration; for example, an insolublesalt of the active compound, such as the decanoate salt, may be adaptedto provide a depot preparation for intramuscular injection.

The pharmaceutical compositions herein will contain, per dosage unit,e.g., tablet, capsule, powder, injection, teaspoonful, suppository andthe like, an amount of the active ingredient necessary to deliver aneffective dose as described above. For example, the pharmaceuticalcompositions herein can contain, per unit dosage unit, from about 25 toabout 400 mg of the active ingredient. Preferably, the range is fromabout 50 to about 200 mg of the active ingredient.

In some embodiments of the present invention carbamate compoundssuitable for use in the practice of this invention will be administeredeither singly or concomitantly with at least one or more other compoundsor therapeutic agents. In these embodiments, the present inventionprovides methods to treat bipolar disorder and modification of symptomsassociated with bipolar disorder m a patient The method includes thestep of administering to a patient in need of treatment, an effectiveamount of one of the carbamate compounds disclosed herein in combinationwith an effective amount of one or more other compounds or therapeuticagents.

It is understood that substituents and substitution patterns on thecompounds of the present invention can be selected by one of ordinaryskill in the art to provide compounds that are chemically stable andthat can be readily synthesized by techniques known in the art as well,as the methods provided herein.

The present invention includes the use of isolated enantiomers ofFormula 1. In one preferred embodiment, a pharmaceutical compositioncomprising the isolated S-enantiomer of Formula 1 is used to providebipolar disorder treatment in a subject. In another preferredembodiment, a pharmaceutical composition comprising the isolatedR-enantiomer of Formula 1 is used to provide bipolar disorder treatmenta subject.

The present invention also includes the use of mixtures of enantiomersof Formula 1. In one aspect of the present invention, one enantiomerwill predominate. An enantiomer that predominates in the mixture is onethat is present in the mixture in, an amount greater than any of theother enantiomers present in the mixture, e.g., in an amount greaterthan 50%. In one aspect, one enantiomer will predominate to the extentof 90% or to the extent of 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% orgreater. In one preferred embodiment, the enantiomer that predominatesin a composition comprising a compound of Formula 1 is the S-enantiomerof Formula 1.

The present invention provides methods of using enantiorners andenantiomeric mixtures of compounds represented by Formula 1, to treatbipolar disorder. A carbamate enantiomer of Formula 1 contains anasymmetric chiral carbon at the benzylic position, which is the secondaliphatic carbon adjacent to the phenyl ring.

An enantiomer that is isolated is one that is substantially free of thecorresponding enantiomer. Thus, an isolated enantiomer refers to acompound that is separated via separation techniques or prepared free ofthe corresponding enantiomer. The term “substantially free,” as usedherein, means that the compound is made up of a significantly greaterproportion of one enantiomer. In preferred embodiments, the compoundincludes at least about 90% by weight of a preferred enantiomer. Inother embodiments of the invention, the compound includes at least about99% by weight of a preferred enantiomer. Preferred enantiomers can beisolated from racemic mixtures by any method known to those skilled in,the art, including high performance liquid chromatography (HPLC) and theformation and crystallization of chiral salts, or preferred enantiomerscan be prepared by methods described herein.

Carbamate Compounds as Pharmaceuticals:

The present invention provides racemic mixtures, enantiomeric mixturesand isolated enantiomers of Formula 1 as pharmaceuticals: The carbamatecompounds are formulated as pharmaceuticals to provide anti-bipolardisorder action in a subject.

In general, the carbamate compounds of the present invention can beadministered as pharmaceutical compositions by any method known in theart for administering therapeutic drugs including oral, buccal, topical,systemic (e.g., transdermal, intranasal, or by suppository), orparenteral (e.g., intramuscular, subcutaneous, or intravenousinjection.) Administration of the compounds directly to the nervoussystem can include, for example, administration to intracerebral,intraventricular, intacerebroventricular, intrathecal, intracisternal,intraspinal or peri-spinal routes of administration by delivery viaintracranial or intravertebral needles or catheters with or without pumpdevices.

Compositions can take the form of tablets, pills, capsules, semisolids,powders, sustained release formulations, solutions, suspensions,emulsions, syrups, elixirs, aerosols, or any other appropriatecompositions; and comprise at least one compound of this invention incombination with at least one pharmaceutically acceptable excipient.Suitable excipients are well known to persons of ordinary skill in theart, and they, and the methods of formulating the compositions, can befound in such standard references as Alfonso A R: Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton Pa.,1985, the disclosure of which is incorporated herein by reference in itsentirety and for all purposes. Suitable liquid carriers, especially forinjectable solutions, include water, aqueous saline solution, aqueousdextrose solution, and glycols.

The carbamate compounds can be provided as aqueous suspensions. Aqueoussuspensions of the invention can contain a carbamate compound inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients can include, for example, a suspendingagent, such as sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productor an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethylene oxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensationproduct of ethylene oxide with a partial ester derived from fatty acidand a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).

The aqueous suspension can also contain one or more preservatives suchas ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose, aspartame or saccharin. Formulations can be adjusted forosmolarity.

Oil suspensions for use in the present methods can be formulated bysuspending a carbamate compound in a vegetable oil, such as arachis oil,olive oil, sesame oil or coconut oil, or in a mineral oil such as liquidparaffin; or a mixture of these. The oil suspensions can contain athickening agent, such as beeswax, hard paraffin or cetyl alcohol.Sweetening agents can be added to provide a palatable oral preparation,such as glycerol, sorbitol or sucrose. These formulations can bepreserved'by the addition of an antioxidant such as ascorbic acid. As anexample of an injectable oil vehicle, see Minto, J. Pharmacol. Exp.Ther. 281:93-102, 1997. The pharmaceutical formulations of the inventioncan also be in the form of oil-in-water emulsions. The oily phase can bea vegetable oil or a mineral oil, described above, or a mixture ofthese.

Suitable emulsifying agents include naturally occurring gums, such asgum acacia and gum tragacanth, naturally occurring phosphatides, such assoybean lecithin, esters or partial esters derived from fatty acids andhexitol anhydrides, such as sorbitan mono-oleate, and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan mono-oleate. The emulsion can also containsweetening agents and flavoring agents, as in the formulation of syrupsand elixirs. Such formulations can also contain a demulcent, apreservative, or a coloring agent.

The compound of choice, alone or la combination with other suitablecomponents can be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like.

Formulations of the present invention suitable for parenteraladministration, such as, for example, by intraarticular (in the joints),intravenous, intramuscular, intradermal, intraperitoneal, andsubcutaneous routes, can include aqueous and non-aqueous, isotonicsterile injection solutions, which car contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Among the acceptable vehiclesand solvents that can be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils canconventionally be employed as a solvent or suspending medium. For thispurpose, any bland fixed oil can be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid canlikewise be used in the preparation of injectables. These solutions aresterile and generally free of undesirable matter.

Where the compounds are sufficiently soluble they can be dissolveddirectly in normal saline with or without the use of suitable organicsolvents, such as propylene glycol or polyethylene glycol. Dispersionsof the finely divided compounds can be, made-up in aqueous starch orsodium carboxymethyl cellulose solution, or in suitable oil, such asarachis oil. These formulations can be sterilized by conventional,well-known sterilization techniques. The formulations can containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents. e.g., sodium acetate, sodiumchloride, potassium chloride, calcium chloride, sodium lactate and thelike.

The concentration of a carbamate compound in these formulations can varywidely, and will be selected primarily based on fluid volumes,viscosities, body weight, and the like, in accordance with theparticular mode of administration selected and the, patient's needs. ForIV administration, the formulation can be a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension can be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation can also be a sterile injectablesolution or suspension in a nontoxic parenterally acceptable diluents orsolvent, such as a solution of 1,3-butanediol. The formulations ofcommends can be presented in unit-dose or multi-dose sealed containers,such as ampoules and vials. Injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

A carbamate compound suitable for use in the practice of this inventioncan be arid is preferably administered orally. The amount of a compoundof the present invention in the composition can vary widely depending onthe type of composition, size of a unit dosage, kind of excipients, andother factors well known to those of ordinary skill in the art. Ingeneral, the final composition can comprise, for example, from 0.000001percent by weight (% w) to 50% w of the carbamate compound, preferably0.00001% w to 25% w, with the remainder being the excipient orexcipients.

Pharmaceutical formulations for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical formulations to be formulated in unit dosage forms astablets, pills, powder, dragees, capsules, liquids, lozenges, gels,syrups, slurries, suspensions, etc. suitable for ingestion by thepatient.

Formulations suitable for oral administration can consist of (a) liquidsolution, such as an effective amount of the pharmaceutical formulationsuspended in, a diluents, such as water, saline or polyethyleneglycol(PEG) 400, (b) capsules, sachets or tablets, each containing apredetermined amount of the active ingredient, as liquids, solids,granules or gelatin; (c) suspensions in an appropriate liquid; and (d)suitable emulsions.

Pharmaceutical preparations for oral use can be obtained throughcombination of the compounds of the present invention with a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable additional compounds, ifdesired, to obtain tablets or dragee cores. Suitable solid excipientsare carbohydrate or protein fillers and include, but are not limited tosugars, including lactose, sucrose, mannitol, or sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose such as methylcellulose, hydroxymethyl cellulose, hydroxypropylmethyl-cellulose orsodium carboxymethylcellulose; and gums including arabic and tragacanth;as well as proteins such as gelatin and collagen.

If desired, disintegrating or solubilizing agents can be added, such asthe cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a saltthereof, such as sodium alginate. Tablet forms can include one or moreof lactose, sucrose, mannitol, sorbitol, calcium phosphates, cornstarch, potato starch, microcrystalline cellulose, gelatin, colloidalsilicon dioxide, talc, magnesium stearate, stearic acid, and otherexcipients, colorants, fillers, binders, diluents, buffering agents,moistening agents, preservatives, flavoring agents, dyes, disintegratingagents, and pharmaceutically compatible carriers. Lozenge forms cancomprise the active ingredient in a flavor, e.g., sucrose, as well aspastilles comprising the active ingredient in an inert base, such asgelatin and glycerin or sucrose and acacia emulsions, gels, and the likecontaining, in addition to the active ingredient, carriers known in theart.

The compounds of the present invention can also be administered in theform of suppositories for rectal administration of the drug Theseformulations can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperatures and will therefore melt in the rectumto release the drug. Such materials are cocoa butter and polyethyleneglycols.

The compounds of the present invention can also be administered byintranasal, intraocular, intravaginal, and intrarectal routes includingsuppositories, insufflation, powders and aerosol formulations (forexamples of steroid inhalants, see Rohatagi, J. Clin. Pharmacol.35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol, 75:107-111,1995).

The compounds of the present invention can be delivered transdermally,by a topical route, formulated as applicator sticks, solutions,suspensions, emulsions, gels, creams, ointments, pastes, jellies,paints, powders, and aerosols.

Encapsulating materials can also be employed with the compounds of thepresent invention and the term “composition” can include the activeingredient in combination with an encapsulating material as aformulation, with or without other carriers. For example, the compoundsof the present invention can also be delivered as microspheres for slowrelease in the body. In one embodiment, microspheres can be administeredvia intradermal injection of drug (e.g., mifepristone)-containingmicrospheres, which slowly release subcutaneously (see Rao, J. BiomaterSci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gelformulations (see, e.g., Gao, Pharm. Res. 12:857-863, 1995); or, asmicrospheres for oral administration (see, e.g., Eyles, J. Pharm.Pharmacol. 49:669-674, 1997). Both transdermal and intradermal routesafford constant delivery for weeks or months. Cachets can also be usedin the delivery of the compounds of the present invention.

In another embodiment, the compounds of the present invent on can bedelivered by, the use of liposomes which fuse with the cellular membraneor are endocytosed, i.e., by employing ligands attached to the liposomethat bind to surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the carbamate compound into target cells in vivo. (See, e.g.,Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin.Biotechnol. 6:698-708, 1995; Ostro, Am. Hasp. Pharm. 46:1576-1587,1989).

The pharmaceutical formulations of the invention can be provided as asalt and can be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thatare the corresponding free base forms. In other cases, the preferredpreparation can be a lyophilized powder which can contain, for example,any or all of the following: 1 mM-50 mM histidine, 0.1%-2% sucrose,2%-7% mannitol, at a pH range of 4.5 to 5.5, that is combined withbuffer prior to use.

Pharmaceutically acceptable salts refer to salts that arepharmaceutically acceptable and have the desired pharmacologicalproperties. Such salts include salts that may be formed where acidicprotons present in the compounds are capable of reacting with inorganicor organic bases. Suitable inorganic salts include those formed with thealkali metals, e.g. sodium and potassium, magnesium, calcium, andaluminum. Suitable organic salts include those formed with organic basessuch as the amine bases, e.g. ethanolamine, diethanolamine,triethanolamine, tromethamine, N methylglucamine, and the like.Pharmaceutically acceptable salts can also include acid addition saltsformed from the reaction of amine moieties in the parent compound withinorganic acids (e.g. hydrochloric and hydrobromic acids) and organicacids (e.g. acetic acid, citric acid, maleic acid, and the alkane- andarene-sulfonic acids such as methanesulfonic acid and benzenesulfonicacid). Pharmaceutically acceptable esters include esters formed fromcarboxy, sulfonyloxy, and phosphonoxy groups present in the compounds.When there are two acidic groups present, a pharmaceutically acceptablesalt or ester may be a mono-acid-mono-salt or ester or a di-salt orester; and similarly where there are more than two acidic groupspresent, some or all of such groups can be salified air esterified.

Compounds named in this invention can be present in unsalified orunesterified form, or in salified and/or esterified form, and the namingof such compounds is intended to include both the original (unsalifiedand unesterified) compound and its pharmaceutically acceptable salts andesters. The present invention includes pharmaceutically acceptable saltand ester forms of Formula (1). More than one crystal form of anenantiomer of Formula 1 can exist and as such are also included in thepresent invention.

A pharmaceutical composition of the invention can optionally contain, inaddition to a carbamate compound, at least one other therapeutic agentuseful in the treatment of bipolar disorder. For example, the carbamatecompounds of Formula 1 can be combined physically with other bipolardisorder treatments in fixed dose combinations to simplify theiradministration.

Methods of formulating pharmaceutical compositions have been describedin numerous publications such as Pharmaceutical Dosage Forms: Tablets,Second Edition. Revised and Expanded. Volumes 1-3, edited by Liebermanet al; Pharmaceutical Dosage Forms: Parenteral Medications. Volumes 1-2,edited by Avis et al; and Pharmaceutical Dosage Forms: Disperse Systems.Volumes 1-2, edited by Lieberman et al; published by Marcel Dekker, Inc,the disclosure of which are herein incorporated by reference in theirentireties and for all purposes.

The pharmaceutical compositions are generally formulated as sterile,substantially isotonic and in full compliance with all GoodManufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Dosage Regimens

The present invention provides methods of providing and-bipolar disorderaction in a mammal using carbamate compounds. The amount of thecarbamate compound necessary to reduce or treat bipolar disorder isdefined as a therapeutically or a pharmaceutically effective dose. Thedosage schedule and amounts effective for this use, i.e., the dosing ordosage regimen will depend on a variety of factors including the stageof the disease, the patient's physical status, age and the like. Incalculating the dosage regimen for a patient, the mode of administrationis also taken into account.

A person of ordinary skill in the art will be able without undueexperimentation, having regard to that skill and this disclosure, todetermine a therapeutically effective amount of a particularsubstituted, carbamate compound for practice of this invention (see,e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd,1999, The art, Science and Technology of Pharmaceutical Compounding; andPickar, 1999, Dosage Calculations). A therapeutically effective dose isalso one in which any toxic or detrimental side effects of the activeagent that is outweighed in clinical terms by therapeutically beneficialeffects. It is to be further noted that for each particular subject,specific dosage regimens should be evaluated and adjusted over timeaccording to the individual need and professional judgment of the personadministering or supervising the administration of the compounds.

For treatment purposes, the compositions or compounds disclosed hereincan be administered to the subject in a single bolus delivery, viacontinuous delivery over an extended time period, or in a repeatedadministration protocol (e.g., by an hourly, daily or weekly, repeatedadministration protocol). The pharmaceutical formulations of the presentinvention can be administered, for example, one or more times daily, 3times per week, or weekly. In one embodiment of the present invention,the pharmaceutical formulations of the present invention are orallyadministered once or twice daily.

In this context, a therapeutically effective dosage of the carbamatecompounds can include repeated doses within a prolonged treatmentregimen that will yield clinically significant results to treat bipolardisorder. Determination of effective dosages in this context istypically based on animal model studies followed up by human clinicaltrials and is guided by determining effective dosages and administrationprotocols that significantly reduce the occurrence or seventy oftargeted exposure symptoms or conditions in the subject. Suitable modelsin this regard include, for example, marine, rat, porcine, feline,non-human primate, and other accepted animal model subjects known in theart. Alternatively, effective dosages can be determined using in vitromodels (e.g., immunologic and histopathologic assays). Using suchmodels, only ordinary calculations and adjustments are typicallyrequired to determine an appropriate concentration and dose toadminister a therapeutically effective amount of the biologically activeagent(s) (e.g., amounts that are intranasally effective, transdermallyeffective, intravenously effective, or intramuscularly effective toelicit a desired response).

In an exemplary embodiment of the present invention, unit dosage formsof the compounds are prepared for standard administration regimens. Inthis way, the composition can be subdivided readily into smaller dosesat the physician's direction. For example, unit dosages can be made upin packeted powders, vials or ampoules and preferably in capsule ortablet form.

The active compound present in these unit dosage forms of thecomposition can be present in an amount of, for example, from about 10mg to about one gram or more, for single or multiple dailyadministration, according to the particular need of the patient Byinitiating the treatment regimen with a minimal daily dose of about onegram, the blood levels of the carbamate compounds can be used todetermine whether a larger or smaller dose is indicated.

Effective administration of the carbamate compounds of this inventioncan be administered, for example, at an oral or parenteral dose of fromabout 0.01 mg/kg/dose to about 150 mg/kg/dose. Preferably,administration will be from about 0,1 mg/kg/dose to about 25 mg/kg/dose,more preferably from about 0.2 to about 18 mg/kg/dose. Therefore, thetherapeutically effective amount of the active ingredient contained perdosage unit as described herein can be, for example, from about 1 mg/dayto about 7000 mg/day for a subject having, for example, an averageweight of 70 kg.

The methods of this invention also provide for kits for use in providingtreatment of bipolar disorder. After a pharmaceutical compositioncomprising one or more carbamate compounds of this invention, with thepossible addition of one or more other compounds of therapeutic benefit,has been formulated in a suitable carrier, it can be placed in anappropriate container and labeled for providing bipolar disordertreatment. Additionally, another pharmaceutical comprising at least oneother therapeutic agent useful in the bipolar disorder treatment can beplaced in the container as well and labeled for treatment of theindicated disease. Such labeling can include, for example, instructionsconcerning the amount, frequency and method of administration of eachpharmaceutical.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and may be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation. The following examples are provided toillustrate specific aspects of the invention and are not meant to belimitations.

A better understanding of the present invention may be obtained in lightof the following examples that are set forth to illustrate, but are notto be construed to limit, the present invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the influence of the tested compound on spontaneousactivity (distance traveled) in mice.

EXAMPLE 1

O-carbamoyl-(D)-phenylalaninol (hereinafter, referred to ‘testcompound’) was tested for the effects on the forced swimming test, ananimal model of depression, in both mice and rats. After single doses ofthe test compound the mean duration of immobility was reduced with anED₅₀ (median effective dose) of 16.6 mg/kg PO (per os) in mice and 18.5mg/kg PO in rats. The test compound was even more potent after multipledosing in mice with an ED₅₀ of 5.5 mg/kg PO. These data suggest that thetest compound shows antidepressant properties.

(Methods)

Male CD-1 mice (16-24 g) and male Wistar rats (90-125 g) were utilizedin these experiments. The test compound (10, 15 and 30 mg/kg) wasdissolved in physiological saline (0.9%) and administered orally in avolume of 1 mL/100 g body weight.

Mice and rats were placed in glass cylinders of 1000 ml beakers (height14 cm, diameter 11.5 cm) and 4000 ml beakers (height 24.5 cm, diameter18.0 cm), respectively, containing water (25 degrees Celsius) up to aheight of 9.0 cm for mice and 19.0 cm for rats. Each mouse or rat wasplaced in the glass cylinder and allowed to swim for 2 minutes,following which, they were observed for a period of 4 minutes for signsof immobility. Immobility was defined as lack of movement, such asfloating in the water with little or no movement of hind legs. Durationof immobility was timed with a stopwatch and recorded. In someexperiments, mice or rats were allowed to swim for 6 or 10 minutes,respectively, one day prior to the forced swimming experiment.

In the single dose test, mice or rats were given test compound or 0.9%NaCl and placed in glass cylinders 1 hour or 4 hours post-treatment,respectively. In the multiple dose experiments, mice were dosed twice aday for 3 days and given an additional dose on Day 4. In addition, themice were placed in the glass cylinders containing water at 25 degreesCelsius and allowed to swim for six minutes on Day 3. Statisticalevaluation was performed using a computer program (The PharmacologicalCalculation System of Tallarida and Murray (#425475-04-7-1992)) based onprobit analysis. Statistical significance was determined using Student'st-test at a P value of <0.05.

(Results)

The test compound, administered in a single dose, to mice reduced meanduration of immobility in a dose-dependent manner for doses of 10, 15and 30 mg/kg PO. 10 mg/kg of the test compound reduced the mean durationof immobility to 101 sec compared to 131 sec for control (with 0.9% NaCLonly). Doses of 15 and 30 mg/kg produced significant reductions of meanimmobility:time from 154 sec (control) to 80 sec and from 132 sec(control) to 30 see, respectively. The ED₅₀ value (50% reduction in meanimmobility time) for the test compound was 16.6 mg/kg.

The test compound, after multiple dosing, to mice reduced mean durationof immobility in a dose-dependent manner for doses of 3, 5 and 8 mg/kgPO. At 3 mg/kg of test compound, mean duration of immobility was reducedto 63 sec from 85 sec for control. Doses of 5 and 8 mg/kg producedsignificant reductions of mean immobility time from 136 sec (control) to73 sec and from 114 sec (control) to 39 sec, respectively. The ED₅₀value for the test compound was 5.5 mg/kg PO.

In rats, the test compound administered at 30 mg/kg significantlyreduced mean duration of immobility from 38 sec (control) to 9 sec at 4hours post-treatment. Doses of test compound at 10 and 15 mg/kg alsoreduced duration of immobility from 74 sec (control) to 62 sec and 65sec (control) to 39 sec, respectively, but these differences were notstatistically significant. The ED₅₀ was 18.5 mg/kg PO which is similarto the ED₅₀ value in mice above.

Mice administered antidepressant compounds show reduction of the meanduration of immobility compared to control as measured in the forcedswim test. Thus, compounds that are active in the mouse forced swim testmay show antidepressant propertied and could alleviate the depressivesymptoms of bipolar disorder.

EXAMPLE 2

The test compound was tested for binding to the dopamine, norepinephrineand serotonin transporters and for the effects on dopamine,norepinephrine and serotonin reuptake. The test compound -showed weakbinding to the dopamine and norepinephrine transporter and weak effectson dopamine and norepinephrine reuptake compared to cocaine.

(Methods)

The test compound was weighed and dissolved in DMSO (dimethyl sulfoxide)to make a 10 or 100 mM stock solution. An initial dilution to 50 or 508μM in assay buffer for binding, or to 1 or 10 mM in assay buffer foruptake, was made. Subsequent dilutions were made with assay buffersupplemented with DMSO, maintaining a final concentration of 0.1% DMSO.Pipetting was conducted using a Biomek 2000 robotic workstation. Theconcentrations of the test compounds are shown in following Table 1.

TABLE 1 Concentrations of Test Compound tested Concentration Assay RangeBinding hDAT (human dopamine transporter) 21.6 nM-100 μM hSERT (humanserotonin transporter) 21.6 nM 100 μM hNET (human norepinephrinetransporter) 21.6 nM-10 μM Uptake hDAT (human dopamine transporter) 31.6nM-10 μM hSERT (human serotonin transporter) 31.6 nM-100 μM hNET (humannorepinephrine transporter) 31.6 nM-100 μM

Inhibition of Radioligand Binding of [¹²⁵I]RTI-55 to hDAT, hSERT or hNETin Clonal Cells:

Cell preparation: HEK293 cells (American Type Culture Collection, ATCC)expressing hDAT, hSERT or hNET inserts are grown to 80% confluence on150 mm diameter tissue culture dishes in a humidified 10% CO₂environment at 37° C. and served as the tissue source. HEK-hDAT andHEK-hSERT cells were incubated in Dulbecco's modified Eagle's mediumsupplemented with 5% fetal bovine serum, 5% calf bovine serum, 0.05 Upenicillin/streptomycin and puromycin (2 μg/mL). HEK-hNET cells wereincubated in Dulbecco's modified Eagle's medium supplemented with 10%fetal bovine serum, 0.05 U penicillin/streptomycin and geneticin (300μg/mL). Cell membranes are prepared as follows. Medium is poured off theplate, and the plate is washed with 10 ml of calcium- and magnesium-freephosphate-buffered saline. Lysis buffer (10 ml; 2 mM HEPES with 1 mMEDTA) is added. After 10 min, cells are scraped from plates, poured intocentrifuge tubes, and centrifuged 30,000×g for 20 min. The supernatantfluid is removed, and the pellet is resuspended in 12-32 ml of 0.32 Msucrose using a Polytron at setting 7 for 10 sec. The resuspensionvolume depends on the density of binding sites within a cell line and ischosen to reflect binding of 10% or less of the total radioactivity.

Assay conditions: Each assay tube contains 50 μl of membrane preparation(about 10-15 μg of protein) prepared as above, 25 μl of test compound,compound used to define non-specific binding (mazindol or imipramine),or buffer (Krebs-HEPES, pH 7.4; 122 mM NaCl, 2.5 mM CaCl2, 1.2 mM MgS04,10 μM pargyline, 100 μM tropolone, 0.2% glucose and 0.02% ascorbic acid,buffered with 25 mM HEPES), 25 μl of [¹²⁵I]RTI-55((−)-2β-Carbomethoxy-3β-(4-iodophenyl)tropane, iornetopane, 40-80 pMfinal concentration) and additional buffer (Krebs-HEPES) sufficient tobring up the final volume to 250 μl. Membranes are preincubated with thetest compound for 10 min at 25° C. prior to the addition of the[¹²⁵I]RTI-55, The assay tubes are incubated at 25° C. for 90 min.Binding is terminated by filtration over GF/C filters using a Tomtec96-well cell harvester. Filters are washed for six seconds with ice-coldsaline. Scintillation fluid is added to each square and radioactivityremaining on the filter is determined using a Wallac μ- or beta-platereader. Specific binding is defined as the difference in bindingobserved in the presence and absence of 5 μM mazindol (HEK-hDAT andHEK-hNET) or 5 μM imipramine (HEK-hSERT). Two or three independentcompetition experiments arc conducted with duplicate detenninations.GraphPAD Prism is used to analyze the ensuing data, with IC₅₀ valuesconvened to K_(i) values using the Cheng-Prusoff equation(K_(i)=IC₅₀/(1+([RTI-55]/K_(d)·RTI-55))).

Filtration Assay for Inhibition of [³H]Neurotransmitter Uptake in HEK293Cells Expressing Recombinant Biogenic Amine Transporters:

Cell preparation: Cells are grown to confluence as, described above. Themedium is removed, and cells are washed twice with phosphate bufferedsaline (PBS) at room temperature. Following the addition of 3 mlKrebs-HEPES buffer, the plates are warmed in a 25° C. water bath for 5min. The cells are gently scraped and then triturated with a pipette.Cells from multiple plates are combined. One plate provides enough cellsfor 48 wells, which is required to generate data on two complete curvesfor the test compound.

Uptake inhibition assay conditions: The assay is conducted in 96 1-mlvials. Krebs-HEPES (350 μl) and test compound, compounds used to definenon-specific uptake, or buffer (50 μl) are added to vials and placed ina 25° C. water bath. Specific uptake is defined as the difference inuptake observed in the presence and absence of 5 μM mazindol (HEK-hDATand HEK-hNET) or 5 μM imipramine (HEK-hSERT). Cells (50 μl) are addedand preincubated with the test compound for 10 min. The assay isinitiated by the addition of [³H]dopamine, [³H]serotonin, or[³H]norepinephrine (50 μl, 20 nM final concentration). Filtrationthrough Whatman GF/C filters presoaked in 0.05% polyethylenimine is usedto terminate uptake after 10 min. The IC₅₀s are calculated applying theGraphPAD Prism program to triplicate curves made up of 6 drugconcentrations each. Two or three independent determinations of eachcurve are made.

(Results)

The test compound was tested for its effects on radioligand([¹²⁵I]RTI-55) binding to and [³H]dopamine uptake by HEK cellsexpressing eDNA for the human dopamine transporter (HEK-hDAT cells), itseffects on radioligand ([¹²⁵I]RTI-55) binding and [³H]serotonin uptakeby HEK cells expressing eDNA for the human serotonin transporter(HEK-hSERT cells), and its effects on radioligand ([¹²⁵I]RTI-55) bindingand [³H]norepinephrine uptake by HEK cells expressing eDNA for the humannorepinephrine transporter (HEK-hNET cells).

In HEK-hDAT cells, the affinity of the compound for the binding site waslower than the affinity of cocaine, the standard compound, for the samesite(s). The K_(i) value for the displacement of [¹²⁵I]RTI-55 by thetest compound was 14,200 nM, and the K_(i) value for cocainedisplacement of [¹²⁵I]RTI-55 binding was 236 nM. In the uptake assaystest compound was less potent at blocking the uptake of [³H]dopamine,with an IC₅₀ value of 2900 nM, as compared to the potency of cocaine(IC₅₀=385 nM). A Hill coefficient other than one suggests complexinteractions with binding or uptake sites.

In HEK-hSERT cells, the affinity of the compound for the binding sitewas lower than the affinity of cocaine, the standard compound, for thesame site(s). The K_(i) value for the displacement of [¹²⁵I]RTI-55 bytest compound was 81,500 nM, and the K_(i) value for cocainedisplacement, of [¹²⁵I]RTI-55 binding was 361 nM. In the uptake assays31,827 was less potent at blocking the uptake of [3H]serotonin, with anIC₅₀ value greater than 100 μM, as compared to the potency of cocaine(IC50=355 nM).

In HEK-hNET cells, the affinity of the test compound for the bindingsite was lower than the affinity of cocaine, the standard compound, forthe same site(s). The K_(i) value for the displacement of [¹²⁵I]RTI-55test compound was 3700 nM, and the K_(i) value for cocaine displacementof [¹²⁵I]RTI-55 binding was 505 nM. In the uptake assays test compoundwas less potent at blocking the uptake of [³H]norepinephrine, with anIC₅₀ value of 4400 nM, as compared to the potency of cocaine (IC₅₀=194nM). The obtained results are shown in following Table 2:

TABLE 2 Effects of test compound on HEK-hDAT, HEK-hSERT and HEK-hNETcells Test Compound Cocaine HEK-hDAT cells [¹²⁵I]RTI-55 Binding K_(i)(nM) 14,200 ± 3,500 236 ± 58 Hill coefficient −0.77 ± 0.12 −0.83 ± 0.04[³H]Dopamine Uptake IC₅₀ (nM) 2900 ± 920 385 ± 54 HEK-hSERT cells[¹²⁵I]RTI-55 Binding K_(i) (nM) 81,500 ± 2,900 361 ± 65 Hill coefficient−2.28 ± 0.05 −0.77 ± 0.04 [³H]Serotonin Uptake IC₅₀ (nM) >100 μM 355 ±39 HEK-hNET cells [¹²⁵I]RTI-55 Binding K_(i) (nM)  3700 ± 1000 505 ± 67Hill coefficient −1.45 ± 0.34 −0.67 ± 0.07 [³H]NE Uptake IC₅₀ (nM)  4400± 1100 194 ± 29

Numbers represent the means±SEM from at least three independentexperiments, each conducted with duplicate (for binding assays) ortriplicate (for uptake assays) determinations. When the K_(i) or theIC₅₀ for the test compound is greater than 10 μM, only two experimentsare conducted and no standard error is reported.

EXAMPLE 3

The test compound administered at 10, 30 and 100 mg/kg subcutaneously(SC) was assessed to determine the influence on spontaneous activity ofwild-type and homozygous mutant dopamine transporter knockout (KO) mice.The test compound selectively reduced activity of the KO mice in adose-dependent manner suggesting that the test compound was highlyefficacious in depressing hyper motor activity in dopamine transporterKO mice.

(Methods)

Male and female wild-type and homozygous mutant dopamine transporter KOmice (n-10 mice/genotype/agent; produced by m vivo homologousrecombination and bred at Duke University Medical Center, Durham, N.C.)were tested for spontaneous activity in the open field following asingle injection of the vehicle or compound. Mice were placed into theopen field for 30 min and administered SC the vehicle (sterile water), 2mg/kg amphetamine, or three concentrations of the test compound (10, 30,100 mg/kg). All drugs were given in a volume of 5 mL/kg. Animals werereturned to the open field for an additional 90 min. Spontaneousactivity was evaluated in an automated Omnitech Digiscan apparatus(Accuscan Instruments, Columbus, Ohio). Activity was summated at 5 minintervals over the 2 h period of testing. Horizontal activity orlocomotion was measured in terms of the total distance covered in cm,vertical activity or rearing was expressed in terms of the total numbersof vertical beam breaks, and stereotypy was quantified in terms ofrepetitive breaks of a given beam or beams with intervals of less thansec. For the analyses, 10 WT and 10 KO mice were run in each of thetreatment groups with approximately equal numbers of males and femalesassigned to each group. Data were analyzed by the Statistical Packagefor Social Sciences programs (version 11.0 for Windows; SASS Science,Chicago, Ill.). The results for each dependent variable were analyzed byrepeated analyses of variance (RMANOVA) for within subjects effects(group differences over time) and between-subjects effects (tests ofmain effects and interactions). Bonferroni corrected pair-wisecomparisons were used as the post-hoc tests. A p<0.05 was consideredsignificant.

(Results)

Baseline: KO mice showed higher levels of locomotor, rearing andstereotypical activities compared to WT mice.

Drug Treatment: Amphetamine at 2 mg/kg SC increased locomotor, rearingand stereotypical activities in WT mice and decreased them in KO animalsrelative to the respective vehicle controls. The test compound reducedactivities in a dose-dependent fashion and the 100 mg/kg dose suppressedactivities more efficiently than amphetamine. Please see representativeFigure below for the locomotor activity (distance traveled in cm)collapsed over the 90 min post-injection period for Amphetamine (AMPH)and test compound. Rearing and stereotyped behavior showed similarresults.

The obtained results are shown in FIG. 1.

EXAMPLE 4

The effects of the test compound (15-30 mg/kg, IP) on the ability toantagonize aggressive behavior in isolated mice were examined. The testcompound showed an ED₅₀ slightly more potent compared to imipramine inreducing isolation-induced aggression.

(Methods)

Male CD-1 mice (15-20 g) were utilized in this experiment. The testcompound (15, 22 and 30 mg/kg) was dissolved in physiological saline(0.9%) and administered orally (PO) in a volume of 1 mL/100 g bodyweight.

Mice were isolated for up to 8 weeks in individual solid wailed wirecages (24×20×17 cm). The animals were not disturbed except to replenishfood. Aggression was tested by placing an intruder mouse into the homecage of the resident animal. Aggression was characterized by an attackon the “intruder” during a three minute period. For Experimentation,following the demonstration of fighting behavior, each resident mousewas given test compound. Only those resident mice displaying fightingbehavior were selected. Sixty minutes later the “intruder” wasreintroduced into the home cage and the pairs were observed for thepresence or absence of fighting behavior. ED₅₀ values were calculatedfrom the percent inhibition of fighting in computer program hosed onprobit analysis.

(Results)

The table below shows the effects of the test compound at doses of 15,22 and 30 mg/kg on fighting behavior. There was dose-related inhibitionof isolation-induced fighting behavior with an ED₅₀ of 25.9 mg/kg. Incomparison, imipramine (30 mg/kg PO inhibited fighting behavior by 50%.

The obtained results are shown in following Table 3.

TABLE 3 Isolation-induced fighting in mice Dose Inhibition of ED₅₀(mg/kg, PO) Treatment (mg/kg PO) n Fighting (%) (confidence limits) TestCompound 15 18 28 25.9 (17.7-37.9) 22 18 44 30 18 56 Imipramine 15 10 20~30 30 10 50

EXAMPLE 5

The primary objective of this study was to determine the efficacy of 2target doses of test compound (200 and 400 mg/day) in comparison withplacebo during 6 weeks of treatment in adult human subjects withmoderate or severe major depression without psychotic features. Anactive comparator (paroxetine) was <included to assist in,distinguishing a negative study from a failed study. In addition, anexit interview was intended to gather information on unexpected benefitsof the test compound in order to refine the clinical developmentprogram. One or both doses of the test compound demonstratedstatistically significantly greater efficacy than placebo on a broadarray of secondary efficacy variables of mood and well-being, suggestingantidepressant activity for the compound. In addition, positive effectsof the test compound on ratings of physical energy/fitness, reduction insadness or depression and mental energy or motivation.

(Methods)

This was a randomized, double blind, parallel-group, active, and placebocontrolled, multicenter study conducted in the U.S. (23 centers) andCanada (4 centers). There Were 2 phases: a pretreatment phase(screening/washout and a baseline visit) and a 6-week, double-blindtreatment phase. Paroxetine, a positive control, was included toevaluate assay sensitivity. After washout (if needed) of prohibitedsubstances, subjects were randomly assigned (1:1:1:1) to receive thetest compound titrated to a target dose of 200 mg/day or 400 mg/day,matching placebo, or a fixed dose (20 mg/day) of parcixetine. Study drugwas given twice daily for 6 weeks. Efficacy and safety were assessedweekly during the double-blind phase. Subjects completing the studyunderwent an Exit Interview (“Your Health and Well-Being”) and completedan Assessment of Benefits of Clinical-trial Drug-treatment (ABCD)questionnaire (U.S. sites only).

(Results)

The table below summarizes the results for the primary efficacy endpointand many of the principal secondary endpoints for the ITT (LOCF) andper-protocol (LOCF) analysis sets. Neither the 200-mg nor the 400-mgdose of the test compound was statistically significantly superior toplacebo on the primary endpoint, change from baseline at Week 6 in theMADRS total score (ITT [LOCF] analysis set), although paroxetine didachieve statistically significant superiority to placebo, confirmingassay sensitivity. However, 1 or both doses of test compound werestatistically significantly superior to placebo or several key secondaryefficacy variables at Week 6. CGI-I (200 and 400 mg), CGI-S (400 mg),MADRS response (>50% improvement in MADRS total score) (400 mg), and thesum of the apparent and reported sadness items (Items 1 and 2) of theMADRS (200 and 400 mg), suggesting antidepressant activity for thecompound. Statistical significance'was achieved more often in secondaryanalyses (across the [LOCF], per-protocol [LOCF], and ITT [observedcase] analysis sets) with test compound 400 mg, but both test compounddoses achieved statistically significant superiority to placebo in manyanalyses. In a few analyses the 200-mg dose achieved statisticalsignificance when the 400-mg dose did not (1 item of the MADRS, 3 itemsof the CDS-R, 1 CDS-R cluster [ITT (LOCI) analysis set]). Thus, therewas no strong evidence for a dose response relationship for testcompound. Paroxetine was superior to placebo, based on the ITT (LOCF),per-protocol (LOCF), and the ITT (observed case) analysis sets, fornearly all secondary efficacy variables. On the self-rated CDS-R, onlyparoxetine was statistically significantly more effective than placebo(for all analysis sets). Results for test compound on the primaryendpoint varied by sex: men achieved nominally statistically significantsuperiority to placebo at 200 mg, whereas women achieved statisticallysignificant superiority to placebo at 400 mg. From the subject'sperspective, based on the Exit Interview and the questionnaire (ABCD) onthe benefits of treatment in the study (U.S. sites only), improvements mmood and well-being were, frequently experienced in all 4 treatmentgroups.

Paroxetine was the medication subjects preferred to take again, followedclosely by test compound 200 mg. Although there were statisticallysignificant differences between each of the active treatments andplacebo on from 8 to 14 of the 51 ABCD items, there were nostatistically significant differences between paroxetine and testcompound (combined), or between test compound 400 mg and test compound200 mg for any of the items on the ABCD questionnaire. The obtainedresults are shown in following Table 4.

TABLE 4 Selected Efficacy Results (Intent-to-Treat [LOCF] andPer-Protocol [LOCF] Analysis Sets) Mean Result TC TC p-Values VersusPlacebo Placebo 200 mg 400 mg Paroxetine TC TC Endpoint_(a) (N = 117) (N= 115) (N = 120) (N = 117) 200 mg 400 mg Paroxetine ITT Primary: (LOCF)MADRS total −10.3 −12.1 −12.4 −14.1 0.118_(b) 0.112_(b) 0.001 scoreSecondary CGI-S Score −1.0 −1.2 −1.3 −1.5 0.061 0.035 0.002 CGI-IScore_(c) —_(c) —_(c) —_(c) —_(c) 0.035 0.030 <0.001 MADRS 27% 36% 41%48% 0.107 0.020 0.001 ressponsed rate MADRS 11% 15% 19% 26% 0.358 0.0660.001 remission_(d) rate MADRS −2.6 −3.2 −3.4 −3.9 0.026 0.012 <0.001sadness (Items 1 + 2) MADRS core −6.7 −8.6 −8.7 −9.7 0.024 0.022 0.001mood subscale 3 Atypical −0.2 −0.4 −0.8 −0.6 0.834 0.078 0.762 items ofHAM-D 31 Per- MADRS total −10.7 −14.8 −15.0 −17.1 0.006_(b) 0.010_(b)<0.001 Protocol score (LOCF) CGI-S score −1.1 −1.5 −1.6 −1.9 0.016 0.022<0.001 CGI-I score_(c) —_(c) —_(c) —_(c) —_(c) 0.004 0.025 <0.001 MADRS29% 40% 50% 60% 0.055 0.011 <0.001 response rate MADRS 14% 20% 29% 35%0.206 0.037 0.003 remission_(d) rate CGI-I = Clinical GlobalImpressions—Improvement; CGI-S = Clinical Global Impressions—Severity;HAM-D 31 = 31-Item Hamilton Depression Scale; LOCF = last observationcarried forward; MADRS = Montgomery-Asberg Depression Rating Scale._(a)Change from baseline at Week 6. _(b)These p-values for test compoundversus placebo are adjusted for multiple comparisons using Dunnett'sprocedure. _(c)Week 6 assessment; categorical analysis. _(d)Remissionwas defined as a MADRS total score less than 9.

Results from the U.S. sites of the Exit Interview and the self-ratedarid blinded ABCD questionnaire on the benefits of treatment in thestudy provided a context for interpreting the results of TC-MDD-201 fromthe subject's perspective. The exit interview data indicated thatpositive experiences most frequently included improvements in mood andwell-being in ail 4 treatment groups. This was supported by data fromthe ABCD questionnaire, in which the most improved aspect of healthduring the trial was “reduction in sadness and depression”. Mood,generally speaking, was the first symptom to improve, with improvementnoticed mostly within the first three weeks of receiving studymedication (Exit interview data).

In general, there were few differences between the 4 treatment groupswithin the 51 items on the ABCD questionnaire Those that were apparentwere generally most robust between placebo and the active medications(i.e., “physical energy/fitness”, “reduction in sadness or depression”and “mental energy or motivation”) rather than between the activemedications themselves. In post hoc analyses, statistically significantsuperiority to placebo was observed for test compound 200 nag, testcompound 400 mg, and paroxetine on 14, 8, and 14 items, respectively, ofthe questionnaire. However, there were no statistically significantdifferences between test compound 400 mg and test compound 200nag, orbetween paroxetine and test compound (combined scores for both doses)for any of the 51 items on the ABCD questionnaire.

References Cited

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

The discussion of references herein is intended merely to summarize theassertions made by their authors and no admission is made that anyreference constitutes prior art. Applicants reserve the right tochallenge the accuracy and pertinence of the cited references.

The present invention is not to be limited in terms of the particularembodiments described in this application, which are intended has singleillustrations of individual aspects of the invention. Many modificationsand variations of this invention can be made without departing from itsspirit and scope, as will be apparent to those skilled in the art.Functionally equivalent methods and apparatus within the scope of theinvention, in addition to those enumerated herein will be apparent tothose skilled in the art from the foregoing description. Suchmodifications and variations are intended to fall within the scope ofthe appended claims. The present invention is to be limited only by theterms of the appended claims, along with the full scope of equivalentsto which such claims are entitled.

1. A method of diminishing manic symptoms in a mammal suffering frombipolar disorder, wherein the mammal is exhibiting manic symptoms ofbipolar disorder, comprising the administration of a therapeuticallyeffective amount of a compound having drag structural Formula (1) or apharmaceutically acceptable salt or amide thereof, to a mammal in needof treatment:

wherein, R is selected from the group consisting of hydrogen, loweralkyl of 1 to 8 carbon atoms, halogen selected from F, Cl, Br and I,alkoxy of 1 to 3 carbon atoms, nitro, hydroxy, trifluoromethyl, andthioalkoxy of 1 to 3 carbon atoms; x is an integer at 1 to 3, with theproviso that R may be the same or different when x is 2 or 3; R₁ and R₂can be the same or different from each other and are independentlyselected from the group consisting of hydrogen, lower alkyl of 1 to 8carbon atoms, phenyl, and cycloalkyl of 3 to 7 carbon atoms; or R₁ andR₂ can be joined to form a 5 to 7-membered unsubstituted heterocyclewherein the heterocyclic compound comprises 1 to 2 nitrogen atoms and 0to 1 oxygen atom, and the nitrogen atoms are not directly connected witheach other or with the oxygen atom; to thereby diminish the manicsymptoms in the mammal suffering from bipolar disorder.
 2. The method ofclaim 1, wherein R is hydrogen.
 3. (canceled)
 4. The method of claim 1,wherein the compound having structural Formula (1) is an enantiomersubstantially free of other enantiomers or an enantiomeric mixturewherein one enantiomer of the compound having structural Formula (1)predominates.
 5. The method of claim 4, wherein one enantiomerpredominates to the extent of about 90% or greater.
 6. The method ofclaim 5, wherein one enantiomer predominates to the extent of about 98%or greater.
 7. The method of claim 4, wherein the enantiomer is (S) or(L) enantiomer as represented by Structural Formula (1a):


8. The method of claim 7, wherein one enantiomer predominates to theextent of about 90% or greater.
 9. The method of claim 8, wherein oneenantiomer predominates to the extent of about 98% or greater.
 10. Themethod of claim 7, wherein R is hydrogen.
 11. The method of claim 4,wherein the enantiomer is (R) or (D) enantiomer, as represented byStructural Formula (1b).


12. The method of claim 11, wherein one enantiomer predominates to theextent of about 90% or greater.
 13. The method of claim 12, wherein oneenantiomer predominates to the extent of about 9% or greater.
 14. Themethod of claim 11, wherein R is hydrogen. 15-17. (canceled)